AZD7762 induces CRBN dependent BAG3 degradation through ubiquitin-proteasome pathway.

Protein degraders are currently under rapid development as a promising modality for drug discovery. They are compounds that orchestrate interactions between a target protein and an E3 ubiquitin ligase, prompting intracellular protein degradation through proteasomal pathway. More protein degraders identification will greatly promote the development of this field. BAG3 is widely recognized as an excellent therapeutic target in cancer treatments. Exploring protein degraders that target BAG3 degradation has profound implications. Herein, molecular docking was applied to assess binding energy between 81 clinical phase I kinase inhibitors and BAG3. BAG3 protein and mRNA level were detected by western blot and quantitative real-time PCR. CCK8 assay and colony formation assay were applied to detect the cell viability and proliferation rate. Cell death was accessed using flow cytometry combined with PI and Annexin V double staining. AZD7762, a Chk1 kinase inhibitor, was identified to induce BAG3 degradation in a ubiquitin-proteasome pathway. AZD7762-induced BAG3 degradation was not dependent on Chk1 expression or activity. CRBN, an E3 ligase, was identified to bind to BAG3 and mediated BAG3 ubiquitination in the presence of AZD7762. By targeting Chk1 and BAG3, two ideal therapeutic targets in cancer treatment, AZD7762 would be a powerful chemotherapy agent in the future.

Trim21 modulates endoplasmic reticulum-associated degradation and sensitizes cancer cells to ER stress-induced apoptosis by inhibiting VCP/Npl4/UFD1 assembly.

Endoplasmic reticulum-associated degradation (ERAD) serves as a crucial quality and quantity control system that removes misfolded or unassembled proteins from the Endoplasmic Reticulum (ER) through the cytoplasmic ubiquitin-proteasome system (UPS), which is critical for cell fate decision. ER stress arises when misfolded proteins accumulated within the ER lumen, potentially leading to cell death via proapoptotic unfolded protein response (UPR). UFD1 in associated with VCP-Npl4, is recognized as a key regulator of protein homeostasis in ERAD. However, the factors that control VCP complex assembly remain unclear. The study elucidates the function of Trim21, an E3 ubiquitin ligase, through its interaction with UFD1, facilitating K27-linkage ubiquitination of UFD1 and inhibiting its incorporation into the VCP complex. This results in the suppression of ERAD substrates degradation and the activation of a proapoptotic unfolded protein response in cancer cells. Additionally, Trim21 over-expression enhances ER stress response and promotes apoptosis upon expose to the ER inducer Tunicamycin. Notably, elevated Trim21 expression correlates with improved overall survival in various tumor types. Overall, the findings highlight the critical role of Trim21 in regulating ERAD progression and cell fate determination in cancer cells through modulation of VCP/Npl4/UFD1 complex assembly.

HMOX1 Promotes Ferroptosis Induced by Erastin in Lens Epithelial Cell through Modulates Fe2+ Production.

PURPOSE: Ferroptosis is defined by the iron-dependent cell death caused by the accumulation of lipid peroxidation. As a major intracellular Fe pools, heme could be metabolized into ferrous iron, carbon monoxide, and biliverdin by Heme oxygenase-1 (HMOX1). Aged human lens epithelium was reported to highly susceptible to ferroptosis, the functional molecular involved in this progress is not explored. Here, we have demonstrated the function of HMOX1 in human lens epithelium during ferroptotic cell death. METHODS: HMOX1 stably expressed cell line was constructed by lentivirus transfection. HMOX1 knock-out cell line was constructed by Crispr-cas9 technology. Protein expression was detected by western blot. Inverted microscope was applied to record the morphological changes among different treatments. CCK8 assay and colony formation assay were applied to detect the cell proliferation rate. Cell death was detected by PI staining. Lipid Peroxidation was detected by Cell malondialdehyde (MDA) assay. Intracellular Ferrous and Ferric ions were determined using an iron assay kit. RESULTS: HMOX1 expression was induced significantly in HLECs under erastin treatment in a time-dependent and dosage-dependent manner. Forced expression of HMOX1 increase the sensitivity of HLECs to erastin treatment. However, knock-out or knock-down of HMOX1 improved the cell viability of HLECs significantly under erastin treatment. Iron liberated from heme by HMOX1 might play pivotal role to improve the sensitivity of HLECs in response to erastin. CONCLUSION: HMOX1 is an essential pro-ferroptosis enzyme which increase the susceptibility of human lens epithelium to erastin. Ferrous iron, a byproduct of heme, might accelerate erastin triggered ferrotosis cell death in human lens epithelium cells.

Triptolide inhibits the progression of Glioblastoma U251 cells via targeting PROX1.

Background: Glioblastoma multiforme (GBM) is the most lethal brain cancer in adults, characterized by rapid growth, extensive invasiveness, and poor prognosis, and there is still a lack of effective treatments. Here, we aimed to explore the role of triptolide (TPL), purified from Tripterygium wilfordii Hook F, on glioblastoma cell growth, apoptosis, proliferation, migration and invasion, as well as potential underlying mechanisms. Methods: The publicly available clinical data of Brain Lower Grade Glioma (LGG) from The Cancer Genome Atlas (TCGA) had been screened to observe PROX1 expression. The Kaplan-Meier analysis was used to analyze the relationship between PROX1 expression and GBM prognosis. CCK8, cell cycle, EDU, apoptosis, wound healing, and transwell assays were performed to detect the effects of TPL on glioblastoma U251 cell viability, cell cycle, proliferation, apoptosis, migration and invasion, respectively. Further, a soft agar colony assay was used to calculate the growth of glioblastoma cells. The qRT-PCR and western blot were conducted to quantify PROX1 mRNA and protein levels. The transcriptional regulation of TPL was detected by Dual luciferase reporter assay. Results: We found that TPL inhibited glioblastoma cell viability, proliferation, cell cycle, migration and invasion, but enhanced apoptosis in a dose-dependent manner. The expression of cell cycle inhibitor, P21, and pro-apoptosis factor, Bax was increased, while invasion-related factors MMP2 and MMP9 were silenced after TPL treatments. Mechanistically, TPL showed transcriptional inhibition of PROX1 appearance. Moreover, ectopic expression of PROX1 partially rescued the effects of TPL on glioblastoma cell viability, proliferation, apoptosis, migration and invasion, and on the expression of cell function-related genes. Conclusion: This study verified that TPL inhibited the progression of glioblastoma cells by transcriptionally depressing the expression of PROX1.

Isolation, purification and antioxidant activities of polysaccharides from Grifola frondosa.

The crude polysaccharides (GFP) were isolated from the fruiting bodies of Grifola frondosa and purified by DEAE cellulose-52 chromatography and Sephadex G-100 size-exclusion chromatography in that order. Three main fractions, GFP-1, GFP-2 and GFP-3, were obtained through the isolation and purification steps. Then the antioxidant activities of these three fractions were investigated in vitro. The results showed that GFP-1, GFP-2 and GFP-3 possessed significant inhibitory effects on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical and superoxide radical; their reducing power, ferrous ions chelating effect and the inhibition ability of the rat liver lipid oxidation where also strong. These results suggest that G. frondosa polysaccharides could be a suitable natural antioxidant and may be the functional foods for humans.